Affinity separation of cells refers to known process techniques where a particular subset of a mixture or population of biological cells are bound to support surfaces by means of ligands with specific affinity to molecules or structures on the cell membranes of the subset. Cells which lack the membrane molecules or structures are not bound to the support surface and can be removed from the population to effect a separation of these cells from the bound cells or vice versa.
Affinity separation processes are commonly used either to eliminate a subset from the mixture of interest (depletion) or to prepare a specific subset of interest from a mixture (positive selection). The depletion process is much simpler because the bound cells are simply discarded leaving the desired cells behind. Positive selection is much more difficult both because the desired cells are bound to the support and must be removed without damaging them and because a certain proportion of the undesirable cells can and do bind nonspecifically to the affinity surface and contaminate the collected cells.
Cell separation techniques have important potential application in cancer therapies, autoimmune disease therapies, and improved diagnostics. For example, cell affinity devices can be used in extracorporeal therapies that may involve the selective isolation, augmentation, and reintroduction to the host of a specific subset population of cells.
Cell affinity techniques have been used widely since Wigzell's description of such a process in 1969 (Wigzell and Andersson J. Exp. Med. 129:23-36, 1969). Cells have been separated using antibodies immobilized to beads (Wigzell and Andersson J. Exp. Med. 129:23-36, 1969), to fibers (Rutishauser et al. Proc. Natl. Acad. Sci. 70, 1973), to petri dishes (Mage et al. J. Immunol. Meth. 15, 1977), and to liquid droplets (U.S. Pat. No. 4,619,904). The separation process basically involves effecting contact between cell mixtures and a ligand-coated support, allowing the cells to bind, and then washing away nonadherent cells.
During the 1970's there were several reports of cell affinity separation techniques for a variety of cells, supports, and ligands (Wysocki and Sato Proc. Natl. Acad. Sci. 75:2844-2848, 1978; Wigzell Scand. J. Immunol. 5:23-30, 1976; Antoine et al. Immunochem. 15, 1987; Edelman and Rutishauser Meth. Enzymol. 34:195-225, 1974). Several patents have issued describing a variety of techniques and devices for affinity cell separations (U.S. Pat. Nos. 4,035,316; 3,970,518; 3,843,324; 4,230,685; 4,363,634). With the exception of panning and certain procedures involving the use of magnetic particles, the techniques have proven difficult to reproduce. Moreover, the many attempts to scale-up these procedures have been very disappointing.
Affinity cell depletion techniques have found some important applications. Researchers prepare specific cell subpopulations for study by systematically depleting a mixture of various subpopulations of cells. For example, Treleavan et al. (Treleaven et al. Lancet 1:70-73, 1984) have demonstrated that the concentration of neuroblastoma cells in a bone marrow preparation can be reduced by a factor of about 10.sup.6 using multiple depletions with antibody-coated magnetic beads.
Two examples of positive selection techniques are those described by Berenson et al. (J. Immunol. Methods 91:11-19, 1986) and by Gaudernack et al. (J. Immunol. Methods 90:179-187, 1986). Berenson et al. bind biotinylated antibodies to target cells and pass them through a column packed with avidin-coated beads, thereby recovering 64% of a population of human bone marrow cells at a final concentration of 73% when the initial concentration was 7%. Gaudernack et al. use antibody-coated magnetic beads to collect a certain subset of T cells. The initial concentration was 30%, and the positively selected population was 96%; the yield is not mentioned. These purities are not adequate for a large number of attractive applications, such as stem cell transplants, or the preparation of subpopulations for cell biology or immunology studies.
Most attempts to scale-up affinity cell separation procedures beyond laboratory scale have involved affinity columns packed with beads coated with antibodies, lectins, or staphylococcus protein A. Solutions containing cell mixtures flow through the column packing and, ideally, the ligands bind the specific cells of interest. In practice, however, a large fraction of the undesired cells bind nonspecifically in the columns such that the purity and yield of the selected cells is disappointingly low. For example, in a recently reported attempt to separate T lymphocytes from peripheral blood lymphocytes using soybean agglutinin-coated Sepharose beads in a column, Hertz et al. found that they could capture 90% of the T lymphocytes, but they also found 80% of the other lymphocytes were nonspecifically bound (Hertz et al. Biotechnology and Bioengineering 27:603-612, 1985). Procedures for cell purification employing the extremely high affinity of biotin and avidin have produced the best large-scale results using packed columns (Berenson et al. J. Cell. Biochem. 10D:239, 1986). Berenson et al. reported the concentration 2.5.times.10.sup.9 target cells from 14% to 73% purity with a yield of 43%.
The most successful approach to large scale cell affinity separations has involved the use of magnetic particles. Magnetic particles have been used clinically to remove neuroblastoma or T cells from bone marrow transplants. 99.97% or more of the unwanted cells are removed from transplants initially containing over 10.sup.10 cells, but over half of the other cells are removed nonspecifically by the process (Vartdal et al. J. Cell. Bioch., Sup. 10D: 252, 1986).
Because of the difficulties in implementing and scaling-up cell affinity separations, this promising technique has found very little practical application. There remains a need to be able to recover cells with higher yields and higher purities.